title: Concerted interaction between origin recognition complex (ORC), nucleosomes and replication origin DNA ensures stable ORC-origin binding.
abstract: Chromosomal replication origins, where DNA replication is initiated, are determined in eukaryotic cells by specific binding of a six-subunit origin recognition complex (ORC). Many biochemical analyses have showed the detailed properties of the ORC-DNA interaction. However, because of the lack of in vitro analysis, the molecular architecture of the ORC-chromatin interaction is unclear. Recently, mainly from in vivo analyses, a role of chromatin in the ORC-origin interaction has been reported, including the existence of a specific pattern of nucleosome positioning around origins and of a specific interaction between chromatin-or core histones-and Orc1, a subunit of ORC. Therefore, to understand how ORC establishes its interaction with origin in vivo, it is essential to know the molecular mechanisms of the ORC-chromatin interaction. Here, we show that ORC purified from yeast binds more stably to origin-containing reconstituted chromatin than to naked DNA and forms a nucleosome-free region at origins. Molecular imaging using atomic force microscopy (AFM) shows that ORC associates with the adjacent nucleosomes and forms a larger complex. Moreover, stable binding of ORC to chromatin requires linker DNA. Thus, ORC establishes its interaction with origin by binding to both nucleosome-free origin DNA and neighboring nucleosomes.
authors.affiliation: Division of Microbial Genetics, National Institute of Genetics, Mishima, 411-8540, Japan.
authors.forename: Kohji
authors.initials: K
authors.lastname: Hizume
authors.forename: Masaru
authors.initials: M
authors.lastname: Yagura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Araki
mesh_terms: D002843:Chromatin / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009707:Nucleosomes / Q000378:metabolism*; D051738:Origin Recognition Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002843:Chromatin; D004271:DNA, Fungal; D009707:Nucleosomes; C497214:ORC1 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins